Intracellular microbial recognition plays a critical role in innate immunity. Small fragments of bacterial peptidoglycan as well as bacterial flagellin are known to stimulate the NOD-like Receptors in mammals. This family of receptors includes both the NOD proteins, which respond to peptidoglycan fragments by activating NF-?B, and the NALP family of proteins, which respond to peptidoglycan, flagellin and other danger signals by activating IL-12 processing and release. Animals deficient in these receptors are hypersusceptible to infection, while humans carrying mutations in NOD2 and NALP3 suffer from autoinflammatory Crohn's disease and Muckle Wells Syndrome. Despite advances, it has not yet been demonstrated that any of these putative intracellular innate immune receptors directly recognize their cognate ligand, nor is it clear how these intracellular receptor transduce signals or how the induced responses contribute to efficient immune protection. In Drosophila, we have recently established that analogous intracellular microbial recognition occurs. Upon injection into flies, small fragments of peptidoglycan are recognized by the intracellular receptor PGRP-LE. Moreover, PGRP-LE directly binds these monomeric fragments of peptidoglycan causing receptor oligomerization. In addition to small fragments of peptidoglycan, PGRP-LE also recognizes cytosolic bacteria, like Listeria monocytogenes, and this recognition is critical to block bacterial growth. These results have led us to hypothesize that PGRP-LE functions as an intracellular receptor capable of recognizing peptidoglycan that enters cells, triggering receptor oligomerization, intracellular signaling and ultimately protective immune responses. We propose a series of 3 Aims that will probe in molecular detail the role of PGRP-LE and intracellular bacterial recognition in producing effective immune responses. Aim 1 will determine which organs, cells and subcellular compartments are involved in the PGRP-LE-mediated response to monomeric peptidoglycan. Aim 2 will characterize the molecular mechanisms of PGRP-LE-mediated signal transduction. The function of ligand-induced receptor oligomerization will be analyzed in vivo; the different responses emanating from intracellular versus cell-surface immune recognition will be probed; and a direct feedback inhibitor of these peptidoglycan binding receptors will be characterized. Aim 3 will analyze the role of PGRP-LE in the response to pathogens that release large quantities of monomeric peptidoglycan. And the role of PGRP-LE in controlling infection by intracellular Listeria monocytogenes will be thoroughly characterized. Autophagy is critical for the control of Listeria and we will determine if PGRP-LE- mediated recognition is required for the induction of this protective response. In people, the innate immune response is absolutely critical for the rapid protection against germs and for the effectiveness of vaccines. In addition, uncontrolled innate immune reactions are often the root cause of auto-inflammatory diseases. Insects, such as the fruit fly, rely entirely on innate immune responses that are very similar to our own innate immune system, and this proposal aims to use the fruit fly, a powerful experimental system, for the study of innate immunity. PUBLIC HEALTH RELEVANCE: In people, the innate immune response is absolutely critical for the rapid protection against germs and for the effectiveness of vaccines. In addition, uncontrolled innate immune reactions are often the root cause of auto-inflammatory diseases. Insects, such as the fruit fly, rely entirely on innate immune responses that are very similar to our own innate immune system, and this proposal aims to use the fruit fly, a powerful experimental system, for the study of innate immunity.